RATIONALE: To determine the negative-ion cleavages from [M–H]– ions of Ser sulfate-containing peptides using experiment and theory in concert. METHODS: Fragmentations were explored using aWaters QTOF2 mass spectrometer in negative-ion electrospray mode, together with calculations at the CAM-B3LYP/6-311++g(d,p) level of theory. RESULTS: Previously, it has been shown that a peptide containing a Tyr sulfate group shows [(M–H)––SO3] as the base
peak. Only a small peak was observed corresponding to HOSO3 – (formed following rearrangement of the sulfate). A Ser sulfate-containing peptide, in contrast, shows pronounced peaks due to cleavage product anions [(M–H)––SO3] and HOSO3
–. Theoretical calculations at the CAM-B3LYP/6-311++g(d,p) level of theory suggest that rearrangement of a Ser sulfate to give C-terminal CO2SO3H is energetically unfavourable in comparison with fragmentation of the intact Ser sulfate to yield [(M–H)––SO3] and HOSO3 –. [(M–H)––H2SO4] anions are not observed in the spectra of peptides containing Ser sulfate, presumably because HOSO3 – is a relatively weak gas-phase base (Δgacid = 1265 kJ mol–1). CONCLUSIONS: Experimental and theoretical data suggest that [(M–H)––SO3] and HOSO3 – product anions (from a peptide with a C-terminal Ser sulfate) are formed from the serine sulfate anion accompanied by specific proton transfer. CID MS/MS/MS data for an [(M–H)––SO3] ion of an underivatised sulfate-containing peptide will normally allow the determination of the amino acid sequence of that peptide. The one case we have studied where that is not the case is LS(SO3H)GDA(OH), where the peptide contains Ser sulfate and Asp, where the diagnostic Asp cleavages are competitive with the Ser sulfate cleavages.